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. 2021 Nov 18;12:6704. doi: 10.1038/s41467-021-26871-y

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — a A schematic diagram shows the fusion of CCDC6 and RET genes in thyroid carcinoma (THCA). b CCDC6-RET fusion events remove the FBW7 degron from the CCDC6 protein, leading to the stabilization of CCDC6-RET fusion proteins. c FBW7 degron of CCDC6 is evolutionarily conserved among species. *Phosphorylation. d Knockout of FBW7 in DLD1 and HCT116 cells leads to accumulation of CCDC6 protein. DLD1 or HCT116 WT and FBW7 deleted cells were harvested, lysed, and immunoblotted for indicated proteins. e Colorectal cancer (CRC) cells with FBW7-mutant exhibited relatively higher protein abundance of CCDC6, compared with those cells with WT FBW7. f CCDC6-RET fusion protein escaped from the recognition by the FBW7 E3 ligase. Cells were transfected with HA-FBW7 together with either GFP-tagged CCDC6 and CCDC6-RET fusion constructs. Cells were treated with 10 μM MG132 for 12 h, followed by harvested, lysed, and immunoprecipitated (IP) with anti-HA. Inputs and immunoprecipitated material were immunoblotted for GFP and HA. g CCDC6-RET fusion protein escaped from FBW7-mediated ubiquitination. Cells were transfected with His-Ub, HA-FBW7 together with either GFP-tagged CCDC6 and CCDC6-RET fusion constructs. Cells were treated with 30 μM MG132 for 6 h, followed by lysed under denaturing conditions. The His-Ub-conjugated proteins were pulled down with Ni-NTA-resin, washed, and immunoblotted for indicated proteins. h Immunoblot analysis for DLD1 stable cell lines that ectopically express either wild-type (WT) CCDC6 or CCDC6-RET fusion protein. i, j CCDC6-RET-expressing cells had greater clonogenicity than WT-CCDC6-expressing cells in vitro. The DLD1 stable cell lines as in h were subjected for colony formation assay (i) and statistical analysis (j). n = 3/group. k, l CCDC6-RET-expressing cells developed larger tumors than WT-CCDC6-expressing cells in vivo. The DLD1 stable cell lines as in h were subjected for mouse xenograft assay (k) and statistical analysis (l). n = 10 mice/group. Data are presented as mean ± SD. **p < 0.01; ***p < 0.001; by two-tailed Student’s t-test. Two independent experiments were performed for d and h. The relevant raw data and uncropped blots are provided in Source Data.